Number and Location of Acetylcholinesterase Molecules at Motor Endplates of the Mouse

Aw, Rogers; Darżynkiewicz, Zbigniew; Barnard, E.A.; Salpeter, MM

doi:10.1038/2101003a0

Cited by 49 publications

(30 citation statements)

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“…23 For the detection of end-plates the most convenient tissue is mouse or rat diaphragm muscle, in which these structures have a characteristic alignment. 29,30 We have therefore exposed the mouse diaphragm muscle to Ph-F, then rinsed it, and examined by fluorescence microscopy. As is evident in Figure 2, the structures in diaphragm muscle that have the characteristic morphological features of the end-plates (arrows) become preferentially labeled with Ph-F.…”

Section: Resultsmentioning

confidence: 99%

Novel Assay Utilizing Fluorochrome-Tagged Physostigmine (Ph-F) to In Situ Detect Active Acetylcholinesterase (AChE) Induced during Apoptosis

Huang¹,

Lee²,

Johnson³

et al. 2004

Cell Cycle

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Section: Resultsmentioning

confidence: 99%

Novel Assay Utilizing Fluorochrome-Tagged Physostigmine (Ph-F) to In Situ Detect Active Acetylcholinesterase (AChE) Induced during Apoptosis

Huang¹,

Lee²,

Johnson³

et al. 2004

Cell Cycle

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“…They were labeled either by: (a) radioactive DFP, to phosphorylate all reactive sites; (b) radioactive DFP followed by 2-PAM, to label all reactive sites except AChe; and (c) nonradioactive DFP, followed by 2-PAM and then by radioactive DFP to label AChe sites only. Conditions for saturation of DFP sites, for minimizing nonspecific binding, and for reactivating with 2-PAM have previously been established and were followed here (31,32). Saturation with DFP was always judged by the absence of cholinesterase reaction products after a 1/2-h staining period using the method of Karnovsky and Roots (24).…”

Section: Methodsmentioning

confidence: 99%

“…This stain was used instead of a Koelle stain previously used by Rogers et al (32) since the latter caused a progressive reduction with time of the sites available for reaction with DFP. After staining, the muscles were washed in phosphate buffer, then incubated with [3zP]DFP (Radiochemical Centre, Amersham, England; sp act 0.2 mCi/ 0.66 mg of DFP), and finally washed in nonradioactive DFP and in buffer, as previously described (31,32). The muscles were teased into individual muscle fibers.…”

Section: Light Microscope Autoradiographymentioning

confidence: 99%

Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography.

Salpeter¹,

Rogers²,

Kasprzak³

et al. 1978

The Journal of Cell Biology

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The distribution of acetylcholinesterase (AChe) in the twitch fibers of the extraocular muscles of the mouse was examined by light and electron microscope autoradiography after labeling with radioactive diisopropyl fluorophosphate (DFP) with, and without, 2-pyridine aldoxime methiodide (2-PAM) reactivation. The values obtained were compared with those previously reported for the diaphragm and sternomastoid muscles. The extraocular muscles were studied because they differ from the other two muscles in that they are among the fastest of the mammalian muscles, yet their endplates have sparse junctional folds. They could thus provide information on the extent to which AChe concentration is an invariant feature of endplate morphology and what, if any, aspects may be related to their fast speed of response.We found, using light microscope autoradiography, that in the twitch fibers of the extraocular muscle, there is an average of 6.4 _ 2.1 • 107 DFP-binding sites per endplate, of which 29% (1.8 • 107) are reactivated by 2-PAM and are thus AChe. The morphology of the extraocular endplates allowed us to conclude, on statistical grounds, that the AChe sites are probably localized not only along the surface area of the postjunctional membrane (PJM) but also along the surface of the presynaptic axonal membrane. Based on this localization, we calculate 7,800 DFP sites and 2,500 2-PAM-reactivated sites//xm 2 of surface area of preand postjunctional membrane. This stacking density of DFP-binding sites per surface area of membrane (probably in the overlying sheets of basal lamina) is very similar to that in the diaphragm and sternomastoid muscles.KEY WORDS acetylcholinesterase extraocular muscle EM autoradiography track autoradiography neuromuscular junctions acetylcholine receptorIn several studies we have assessed the number of esterase sites both at the whole endplate (31, 32) and on the fine structural level (33,34,41). We found that in two different mouse muscles, the diaphragm and sternomastoid, there were about the same number of labeled sites per square micrometer of the folded postjunctional membrane (PJM). These data suggested that the esterases may be present in muscles in some constant 274 J. CELL BIOLOGY (~ The Rockefeller University Press 9

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“…In the present study, the distribution of AChase within the motor, end plate of a vertebrate twitch muscle was evaluated quantitatively by the use of electron microscope radioautography after this enzyme was selectively labeled with DFp-aH. Some of the results of this work are included in a preliminary report (Rogers et al 1966).…”

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confidence: 99%

“…These authors applied light microscope radioautography to the quantitative evaluation of acetylcholinesterase (AChase) at motor end plates using the irreversible enzyme inhibitor diisopropylfluorophosphate-~H (DFP-3H) (Ostrowski et al, 1963;Barnard and Ostrowski, 1964). It has subsequently been shown (Rogers et al, 1966) that some quantitative results reported in these inves-tigations were subject to error. This was probably due to a very high uptake of DFP by muscle mast cells, which were not distinguished from end plates in the unstained material used.…”

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Electron Microscope Radioautography as a Quantitative Tool in Enzyme Cytochemistry

Salpeter

1967

The Journal of Cell Biology

126

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Tritiated diisopropylfluorophosphate (DFP) was used to phosphorylate acetylcholinesterase (AChase) in the motor end plate of mouse sternomastoid muscle, and its distribution within the end plate was evaluated quantitatively by electron microscope radioautography. With the use of emulsion layers whose sensitivity to tritium had been calibrated, the density of AChase in different components of the end plate was calculated. The AChase was primarily localized (85 %) in the junctional fold region. The concentration of AChase there was more than 20,000 active sites per cubic micron of tissue. The resolution of the technique was not sufficient to determine whether there was some AChase in the nerve end bulb; however, if there is any there, the concentration must be less than 10% of that at the junctional fold region.

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Number and Location of Acetylcholinesterase Molecules at Motor Endplates of the Mouse

Cited by 49 publications

References 10 publications

Novel Assay Utilizing Fluorochrome-Tagged Physostigmine (Ph-F) to In Situ Detect Active Acetylcholinesterase (AChE) Induced during Apoptosis

Novel Assay Utilizing Fluorochrome-Tagged Physostigmine (Ph-F) to In Situ Detect Active Acetylcholinesterase (AChE) Induced during Apoptosis

Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography.

Electron Microscope Radioautography as a Quantitative Tool in Enzyme Cytochemistry

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